bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433159; this version posted March 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Short Note Uncovered genetic diversity in Hemidactylus mabouia (Reptilia: Gekkonidae) from Madeira Island reveals uncertain sources of introduction Catarina Rato1*, Beatriz Martins1,2, Ricardo Rocha1,3†, Iolanda Silva-Rocha1† 1 CIBIO, Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus de Vairão, Rua Padre Armando Quintas nº7, Vairão 4485 - 661, Vila do Conde, Portugal. 2 Departamento de Biologia, Faculdade de Ciências da Universi dade do Porto, R. Campo Alegre, s/n, 4169 - 007, Porto, Portugal. 3 CIBIO- InBIO, Research Center in Biodiversity and Genetic Resources, Institute of Agronomy, University of Lisbon, Lisbon, Portugal † These authors have contributed equally to this work * Corresponding author: [email protected] Total number of words in the whole manuscript: 3,274 Total number of words in the abstract: 151 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433159; this version posted March 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 36 Abstract 37 Hemidactylus mabouia is one of the most widely distributed species within its genus. It was 38 first reported to Madeira in 2002 and the first individuals were considered to have originated 39 from Cape Verde. Almost 20 years later, we found that H. mabouia has substantially expanded 40 its distribution and can now be found >10 km away from Funchal, where it was first reported. 41 Based on a 12S phylogenetic analysis and using 29 individuals from Funchal and Câmara de 42 Lobos we found that Madeira actually harbours two distinct lineages of H. mabouia: one 43 exclusively South American and another widespread in America and Africa. However, the lack 44 of genetic diversity typical of this species outside its native range and the obtained phylogenetic 45 pattern prevent us to infer possible introduction routes or sources. Our study emphasizes that 46 authorities should remain vigilant regarding the arrival of other non-natives and act to prevent 47 their establishment as soon as they are detected. 48 49 Keywords: Hemidactylus mabouia, Madeira Island, biological invasions, conservation, 50 phylogenetic analysis 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433159; this version posted March 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 69 The rate of species’ translocation to areas outside their native geographical range is increasing 70 with human globalization, considerably impacting native biotas (Simberloff et al, 2013). 71 Reptiles are particularly prone to biological invasions, either by being one of the most often 72 introduced animal groups, or by being notably sensitive to the impacts of invaders (Kraus, 73 2009). The introduction of species to island ecosystems is particularly problematic, due to the 74 limited capacity of insular native species to compete with newcomers, elude predators and 75 handle diseases, inherent from their evolutionary insular history (Novosolov et al, 2013; 76 Whittaker and Fernández-Palacios, 2007). Hemidactylus Gray, 1845 is one of the most species 77 rich genera within the Gekkonidae family, currently comprising about 164 recognized species 78 (Uetz et al, 2020). These geckos have their native distribution throughout most of tropical Asia 79 and Africa, the more arid regions of northeast Africa and southwest Asia, the Mediterranean 80 region and South America, which they reached by natural transmarine colonization (Kluge, 81 1969). They have recently colonized, both naturally and anthropogenically, other areas of the 82 Americas and the West Indies, Australia and several islands of the Indian, Pacific and Atlantic 83 Oceans (see references in Carranza and Arnold, 2006). Their success as human-assisted 84 colonizers is partly associated with the synanthropic habits of several Hemidactylus species, 85 allied with their relatively small size and cryptic nature (Hoskin, 2011). The tropical house gecko 86 Hemidactylus mabouia is one of the most successful invaders within the genus, and it can now 87 be found in both the Old and New Worlds, as well as in many Atlantic islands, such as Cape 88 Verde (Jesus et al, 2001; although classified later as Hemidactylus mercatorius in Vasconcelos 89 et al, 2013 following Rocha et al, 2010), the Gulf of Guinea Islands (Jesus et al, 2005) and 90 Madeira (Jesus et al, 2002). The successive introductions and absence of genetic differentiation 91 within H. mabouia over its extensive range, precludes the assessment of the ultimate 92 geographic origin of this species (Carranza and Arnold, 2006). Nonetheless, the great genetic 93 diversity observed on Mayotte island in the Comoros, and the relationship to northeast 94 Madagascar populations assigned to H. mercatorius, suggest that the widespread haplotypes 95 originated in East Africa, and from there spread westwards, across tropical Africa reaching 96 several Atlantic islands and large parts of tropical America (Carranza and Arnold, 2006). 97 Additionally, based on the deep divergence observed between African and Malagasy 98 specimens, Vences et al (2004) began to refer to all H. mabouia populations in Madagascar as 99 Hemidactylus mercatorius (Gray, 1842). Moreover, results from Rocha et al (2005) clearly 100 show that some specimens of H. mabouia from the Gulf of Guinea cluster with one H. 101 mercatorius clade from northern Madagascar and the Comoros. Because Boumans et al (2007) 102 argued in favour of the native status of H. mercatorius across Madagascar, Rocha et al (2010) 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433159; this version posted March 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 103 proposed that insular populations of H. mabouia should be recognized as H. mercatorius, and 104 the status of East African populations should remain unchanged as H. mabouia, pending further 105 studies. Clearly, the complex phylogeographical patterns and taxonomy of the putative 106 “species-complex” H. mabouia/mercatorius is far from being understood. 107 In this study, we focus on the genetic affinities of H. mabouia specimens from Madeira Island, 108 in an attempt to infer the geographical origin of the introduced populations. The volcanic 109 archipelago of Madeira is located ca. 520 km from the African coast and is comprised by the 110 islands of Madeira, Porto Santo and Desertas (fig. 1). Almost 20 years ago, Jesus et al (2002) 111 reported the first three specimens in Madeira, captured in the island’s capital Funchal. These 112 animals were assigned as H. mabouia based on their genetic similarity with other sequences 113 available at the time on GenBank (12S and cytb). Through the analysis by eye of the alignment, 114 the authors concluded that the Madeiran specimens were most similar to previously sampled 115 individuals from the Cape Verdean island of São Vicente. Here, apart from collecting 116 specimens from an additional locality in Madeira, the total number of analysed individuals is 117 substantially larger, allowing the detection of possible within island intraspecific genetic 118 variation. Additionally, through a phylogenetic analysis, we use available H. mabouia 119 sequences from the species’ known distribution to infer possible routes and sources of 120 introduction of Madeira’s populations of tropical house geckos. 121 122 A total of 29 individuals assigned in the field to H. mabouia were collected from two distinct localities in Madeira, 123 namely Funchal (N=16) and Câmara de Lobos (N=13). In Câmara de Lobos, some individuals were collected in 124 the city centre, while others captured in a nearby industrial area. 125 Tissue from tail tip muscle was collected from each individual and preserved in 96% ethanol. Genomic DNA was 126 extracted using an Easy-Spin Kit (Citomed). A fragment of the 12S rRNA gene was amplified by Polymerase 127 Chain Reaction (PCR) using the primers L1091 and H1478 published by Kocher et al (1989). Amplification of 128 the 12S mtDNA fragment was carried out in a 25 µl volume, containing 0.16 µM of each primer, 1U of Taq DNA 129 polymerase (MyTaq HS Mix, Bioline), and approximately 100 ng of template DNA. PCR consisted of an initial 130 denaturation at 95°C for 10 min, followed by 40 cycles that included a denaturation step at 95°C for 30 sec, 131 annealing at 54°C for 30 sec and extension at 72°C for 30 sec. A final extension was conducted for 10 min. All 132 successful amplifications were sent to CTM (CIBIO-InBIO) for purification and Sanger sequencing. This same 133 protocol was also employed on two H. mercatorius specimens from Madagascar, with codes ACP1143 134 (Sahamalaza) and ACP2350 (Isalo). 135 A total of 35 12S sequences of H. mabouia were retrieved from GenBank and added to the dataset. Geographic 136 location of each population from Madeira is depicted in fig.